Fluorescent phosphatidylinositol 4,5-bisphosphate derivatives with modified 6-hydroxy group as novel substrates for phospholipase C

Article abstract of DOI:10.1021/bi300637h

The capacity to monitor spatiotemporal activity of phospholipase C (PLC) isozymes with a PLC-selective sensor would dramatically enhance understanding of the physiological function and disease relevance of these signaling proteins. Previous structural and biochemical studies defined critical roles for several of the functional groups of the endogenous substrate of PLC isozymes, phosphatidylinositol 4,5-bisphosphate (PIP₂), indicating that these sites cannot be readily modified without compromising interactions with the lipase active site. However, the role of the 6-hydroxy group of PIP₂ for interaction and hydrolysis by PLC has not been explored, possibly due to challenges in synthesizing 6-hydroxy derivatives. Here, we describe an efficient route for the synthesis of novel, fluorescent PIP₂ derivatives modified at the 6-hydroxy group. Two of these derivatives were used in assays of PLC activity in which the fluorescent PIP₂ substrates were separated from their diacylglycerol products and reaction rates quantified by fluorescence. Both PIP₂ analogues effectively function as substrates of PLC-δ1, and the K_M and V_max values obtained with one of these are similar to those observed with native PIP₂ substrate. These results indicate that the 6-hydroxy group can be modified to develop functional substrates for PLC isozymes, thereby serving as the foundation for further development of PLC-selective sensors.

Full text of DOI:10.1021/bi300637h

Article
pubs.acs.org/biochemistry
Fluorescent Phosphatidylinositol 4,5-Bisphosphate Derivatives with
Modified 6-Hydroxy Group as Novel Substrates for Phospholipase C
Xiaoyang Wang,† Matthew Barrett,‡ John Sondek,‡ T. Kendall Harden,‡ and Qisheng Zhang*,†
†Division of Chemical Biology and Medicinal Chemistry and ‡Department of Pharmacology, University of North Carolina at Chapel
Hill, Chapel Hill, North Carolina 27599, United States
S
* Supporting Information
ABSTRACT: The capacity to monitor spatiotemporal activity of
phospholipase C (PLC) isozymes with a PLC-selective sensor
would dramatically enhance understanding of the physiological
function and disease relevance of these signaling proteins.
Previous structural and biochemical studies defined critical roles
for several of the functional groups of the endogenous substrate
of PLC isozymes, phosphatidylinositol 4,5-bisphosphate (PIP2),
indicating that these sites cannot be readily modified without
compromising interactions with the lipase active site. However,
the role of the 6-hydroxy group of PIP2 for interaction and
hydrolysis by PLC has not been explored, possibly due to
challenges in synthesizing 6-hydroxy derivatives. Here, we
describe an efficient route for the synthesis of novel, fluorescent
PIP2 derivatives modified at the 6-hydroxy group. Two of these
derivatives were used in assays of PLC activity in which the fluorescent PIP2 substrates were separated from their diacylglycerol
products and reaction rates quantified by fluorescence. Both PIP2 analogues effectively function as substrates of PLC-δ1, and the
KM and Vmax values obtained with one of these are similar to those observed with native PIP2 substrate. These results indicate that
the 6-hydroxy group can be modified to develop functional substrates for PLC isozymes, thereby serving as the foundation for
further development of PLC-selective sensors.
he phospholipase C (PLC) family of enzymes catalyze the
hydrolysis of membrane-bound phosphatidylinositol 4,5-
GTPases.18 However, when, where, and how various PLC
isoforms are activated under different extracellular stimuli are
still not well understood. This is partly due to the lack of
methods to monitor the spatiotemporal activities of cellular
PLCs.
Standard, radioisotope-based assays are discontinuous and
cannot be used to monitor the real-time dynamics of PLC
activity in cells. Cell permeable dyes that increase in
fluorescence upon binding calcium are also routinely used as
complementary methods to monitor PLC activity. However,
although such assays are simple and throughput is high, they
are not a direct measure of PLC activity and often generate
confounding data since other factors also contribute to
intracellular calcium concentration.
With the long-term goal of spatiotemporal monitoring of
inositol lipid signaling, we initiated studies to develop small
molecule sensors of PLCs. Previous kinetic studies demon-
strated that the 2-hydroxy (2-OH) in PIP2 is essential19−21 for
its hydrolysis by PLCs and that 3-phosphoinositides are not
PLC substrates.22 Similarly, removal of the phosphate group
from the 4- or 5-OH positions resulted in derivatives that are
T
bisphosphate (PIP2) to form the second messengers,
diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3).1
Diacylglycerol activates protein kinase C (PKC) while IP3
activates calcium channels leading to the release of Ca2+ from
endothelial reticulum to cytosol.2 In addition, PLC action also
regulates local PIP2 pools leading to changes in subcellular
localization and/or function of a broad range of PIP2-
interacting proteins, including numerous ion channels.
Consequently, PLCs are essential signaling proteins that
regulate diverse cellular processes, including proliferation3 and
differentiation,4 vasculogenesis,5 and fertilization.6 Aberrant
regulation of PLCs has been implicated in diseases including
cancer,7−13 heart diseases,14 and neuropathic pain.15
The 13 mammalian PLC isozymes are broadly grouped into
six families (-β, -γ, -δ, -ε, -ζ, -η) based on their sequence
homologies and functions. Various extracellular stimuli activate
different PLC isoforms through distinct mechanisms. For
example, agonists of G-protein coupled receptors (GPCRs)
activate PLC-β isozymes through Gαq- and Gβγ-subunits of
heterotrimeric G proteins, while PLC-γ activity is enhanced
through phosphorylation promoted by receptor and non-
receptor tyrosine kinases. In addition, PLC-ε and certain
members of the PLC-β and PLC-γ subclasses of isozymes are
activated by Ras,16 Rho,17 and Rac subfamilies of small
Received: May 16, 2012
Revised: June 14, 2012
Published: June 15, 2012
© 2012 American Chemical Society
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poor substrates for PLCs. Furthermore, the stereochemistry of
the D-myo-insoitol headgroup, but not the diacylglycerol side
chain, was shown to be critical for effective PLC-mediated
catalysis.23 Finally, alkyl substitutions at the sn-1 and sn-2
positions resulted in PIP2 molecules that still function as PLC
substrates.24 In general, the longer the side chain, the more
efficient the corresponding PIP2 derivative was for PLC-
promoted hydrolysis.25 These results are supported by the
crystal structure26,27 of the catalytic domain of PLC-δ1 in
complex with IP3; the 1-, 4-, and 5-phosphates and the 2- and 3-
hydroxyls of IP3 interact with PLC-δ1, and mutation of these
interacting residues greatly reduced the capacity of PLC-δ1 to
hydrolyze PIP2.28 Consequently, modifications of PIP2 to
develop new PLC substrates have avoided targeting the inositol
headgroup and have instead focused on the diacylglycerol side
chain. For example, we29 and others30−33 have developed
fluorogenic PLC reporters with modifications at the 1-
phosphate to monitor PLC activity in vitro. However, these
systems are unlikely to be optimal for studies in live cells since
potential spatial information on PLC activity will be lost upon
cleavage of the fluorophore from the PIP2 derivative.
that contains purified, full-length PLC-δ1 (0.05 or 0.1 ng/μL,
final concentration) and BSA (1 mg/mL), and the reaction
mixtures were incubated at 25 °C. At indicated time points,
samples were taken out of the reaction mixtures with a
multichannel pipet (1 μL) and spotted on TLC plates (Merck,
Silica Gel-60). The TLC plates were then developed with
CHCl3:MeOH:H2O (100:20:1) and scanned with a Typhoon
9400 Variable Mode Imager (λex/λem = 488 nm/520 nm). The
fluorescence of DAG and PIP2 derivatives on the TLC plate
was quantified with ImageQuant software (V.5.0).
Fluorescence Calibration and Reaction Rate Calculation.
To a set of water solutions (20 μL) containing various
concentrations (432, 324, 216, 108, 54, 32, 24, and 12 μM) of
PIP2 derivative 1 was added the 6X buffer solution (5 μL) as
defined previously and a PLC solution (5 μL) containing
purified, full-length PLC-δ1 (10 ng/μL, final concentration)
and BSA (1 mg/mL). The reaction mixtures were incubated at
25 °C for 2 h to completely convert probe 1 to DAG. As a
control, the reactions were also carried out in the presence of
BSA (PLC free). These two sets of reaction mixtures were
spotted on TLC plates which were subsequently developed and
the fluorescence was quantified. The fluorescence was plotted
against concentration for probe 1 or DAG. The slope of the
plot for probe 1 was 0.66-fold of that for DAG. Consequently,
the amount of DAG that was generated from the reaction was
calculated with the formula
To our knowledge, selective modification of the 6-OH has
not been explored despite extensive previous efforts synthesiz-
ing PIP2 analogues for novel functions and assays. The structure
of PLC-δ1 bound to IP3 highlights a lack of direct interaction
between the lipase active site and the 6-OH group, indicating
that the 6-OH is solvent exposed and its modification in PIP2 is
likely tolerated within the active site of PLCs. Consequently, we
designed and synthesized three PIP2 derivatives (Figure 1) and
DAG (pmol) = initial PIP concentration (μM)
2
× volume (μL) × (0.66 × a)
/(0.66 × a + b)
where a = fluorescence of DAG and b = fluorescence of PIP2.
Kinetic Studies of Endogenous PIP2. A mixture of PIP2
(Avanti Polar Lipids) and 30 000 cpm of [3H]PIP2 (Perkin-
Elmer) was dried under a stream of nitrogen and resuspended
in assay buffer that contains HEPES (50 mM, pH 7.2), KCl (70
mM), EGTA (3 mM), DTT (2 mM), CaCl2 (2.96 mM), 0.5%
cholate, and BSA (0.2 mg/mL). The resulting lipid stock was
diluted to obtain final assay conditions with either 216, 144, 72,
36, 24, 16, 8, or 4 μM PIP2 in assay buffer (50 μL, final
volume). Assays were initiated by the addition of purified, full-
length PLC-δ1 (0.075 ng) in assay buffer (10 μL). After
incubation at 25 °C for 10 min, reactions were stopped by the
addition of 10% (v/v) trichloroacetic acid (200 μL) and 10
mg/mL BSA (100 μL) to precipitate proteins and uncleaved
lipids. Centrifugation of the reaction mixture isolated soluble
[3H] IP3, which was quantified using liquid scintillation
counting.
Figure 1. Chemical structures of the PIP2 derivatives 1−3 as PLC
substrates.
investigated their capacity to be hydrolyzed by PLC-δ1. Probe 1
has a free 6-OH identical to endogenous PIP2 while probes 2
and 3 have modified 6-OH groups, with a propyl modification
in 2 and an extended hydroxy in 3. The incorporation of a
fluorescent group at the sn-1 position of the DAG side chain
provides a sensitive detection of both the substrate and the
product by fluorescence, thus avoiding the use of radioactive
materials and providing a potential avenue for the use of real-
time monitoring of phospholipase activity in cells with high
spatiotemporal resolution and high throughput.
RESULTS AND DISCUSSION
■
1. Synthesis of the Fluorescent Probes. Fluorescent
PIP2 derivatives with free 6-OH such as 1 have been used to
image cellular PIP2 localization34 and as probes for metabolic
enzymes such as phosphoinositide 3-kinase (PI3K).35 Con-
sequently, the synthesis of 1 follows the literature proce-
dure.36−38 In contrast, PIP2 analogues with 6-OH selectively
modified have not been reported and their syntheses are
challenging. To synthesize the probe 2 (Scheme 1) with 6-OH
group protected as its propyl ether, we started with the known
inositol derivative 4. Alkylation of 4 with allyl bromide followed
by deacetalization with trifluoroacetic acid (TFA) generated 5
in 74% yield. The diol in 5 was subsequently protected as the
EXPERIMENTAL PROCEDURES
■
Kinetic Studies of Probes 1−3. Enzymatic Reactions.
The fluorescent PIP2 derivatives were dissolved in water to
make working solutions at 432, 324, 216, 108, 54, 32, 24, or 12
μM. To these solutions (20 μL) were added a 6X buffer (5 μL)
that contains HEPES (300 mM, pH 7.2), KCl (420 mM),
CaCl2 (17.8 mM), EGTA (18 mM), DTT (12 mM), and 3%
cholate. The reaction was initiated by adding a solution (5 μL)
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Scheme 1. Synthesis of Probe 2
Scheme 2. Synthesis of Probe 3
corresponding methoxymethyl (MOM) ethers. Next, the
tetraisopropyldisiloxane (TIPDS) group was removed by
treating with hydrofluoride (HF) in CHCl3/CH3CN. The
resulting hydroxy groups were phosphorylated to form 7 in a
two-step sequence: first reacted with dibenzyl diisopropylphos-
phoramidite in the presence of tetrazole and then oxidized by
3-chloroperoxybenzoic acid (mCPBA). The tert-butyldiphenyl-
silyl (TBDPS) protection was removed by tetrabutylammo-
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Figure 2. Validation of the TLC-fluorescence assay. (A) Probes 1−3 are cleaved by PLC-δ1 to form inositol trisphosphate and diacylglycerol (DAG)
derivatives. (B) The separation of PIP2 derivatives 1−3 and their corresponding DAGs on thin-layer chromatography (TLC). The reaction mixture
(1 μL) was spotted on the TLC plate and separated by CHCl3:MeOH:H2O (100:20:1). The fluorescent compounds were detected by a Typhoon
9400 variable mode imager (λex/λem = 488 nm/520 nm). (C) Plot of fluorescence versus concentration of DAG or probe 1.
nium fluoride (TBAF) to form alcohol 8, which was coupled to
the diacylglycerol side chain 9 to form 10 in 90% yield through
phosphorylation followed by oxidation with tert-butyl peroxide
(t-BuOOH). To remove the MOM protective groups,
compound 10 was treated with trimethylsilyl bromide
(TMSBr) in CH2Cl2 followed by methanolysis. The benzyl
protective groups were also partially removed during this
process. Hydrogenolysis completed the deprotection of both
benzyl and carbobenzyloxy (Cbz) groups and reduced the allyl
to propyl group to form 11. Finally, the terminal amine was
acylated with activated fluorescein 12 to generate the
fluorescent PIP2 derivative 2 with 6-OH capped as a propyl
ether. The overall yield of the synthesis was 37%.
To synthesize the probe 3 (Scheme 2), olefin metathesis of 6
with MOM-protected allylic alcohol was carried out. We did
not attempt to quantify the E/Z ratio although the formed
double bond had predominantly the E configuration as judged
by NMR. Instead, the olefin was directly subjected to
hydrogenation to form 13. In a sequence that is analogous to
the synthesis of probe 2, the diol 13 was used to generate probe
3. The overall yield of this sequence was 15%.
2. Thin-Layer Chromatography Coupled with Fluo-
rescence Scanning as a New Assay for PLC Activity. The
classical in-vitro assay of PLC activity requires the use of
radioactive PIP2, particularly tritium-labeled PIP2, as the PLC
substrate. To avoid the use of radioactive PIP2, 31P NMR has
been used to monitor PLC-catalyzed reactions.39 However, the
concentration of PIP2 has to be high (>0.5 mM) to obtain
accurate measurement, and these techniques are not amenable
to large numbers of samples. Existing fluorogenic31,32 and
luminescence-based33 reporters have the advantage of con-
tinuous monitoring of PLC activity with high sensitivity.
However, these reporters typically replace the diacylglycerol
unit connected to the 1-phosphate in PIP2, thereby limiting
their utility to faithfully recapitulate intracellular events. In
contrast, 6-OH analogues provide unexplored potential to
produce useful biosensors of PLC activity in cells.
expected PLC-dependent hydrolysis products (Figure 2A) are
predicted to be readily separable by either TLC or column
chromatography, and subsequent detection by fluorescence
should provide a new assay for PLC activity. To validate this
format, purified PLC-δ1 was used to hydrolyze probe 1. Based
on sequence and structural similarities, the catalytic domain of
PLC-δ1 is representative of the entire family of PLC isozymes.
The enzymology of PLC-δ1 has also been extensively
studied20,26,28,40−42 and benefits from the unique structure of
PLC-δ1 bound to IP3 within its active site. The purified enzyme
was incubated with probe 1 at room temperature for 10 min,
and the mixture was separated on TLC and detected by
fluorescence. Fluorescence at the origin of application
represents probe 1 and a second fluorescent component
appeared as a function of increasing amounts of time during the
initial incubation (Figure 2B). This new component was
confirmed by LC-MS analysis to be the diacylglycerol
derivatives predicted to be generated upon hydrolysis of
probe 1 by PLC-δ1. When probe 1 was incubated with
catalytically inactive PLC-δ1 (E341A),28 no new fluorescent
components were formed (Figure 2B), indicating that the
hydrolysis of 1 is dependent on the lipase activity of PLC-δ1.
Conditions were established to quantify the fluorescent DAG
derivative formed during the reaction. Briefly, the linear plots of
the fluorescence versus concentration for both 1 and the
corresponding DAG product are shown in Figure 2C. The ratio
of the slopes was used as the coefficient to normalize
fluorescence readings for quantifications. Accordingly, 8% of
probe 1 was converted to product in Figure 2B. Under identical
conditions, 3% and 5% of probes 2 and 3 were converted to
product, respectively. Like probe 1, probes 2 and 3 were not
cleaved by catalytically inactive PLC-δ1 (E341A) suggesting
that both are selective substrates for PLC-δ1.
3. Kinetic Studies of Probes 1−3 with PLC-δ1. As
highlighted in Figure 2B, probes 1−3 were cleaved by PLC-δ1
with different efficiencies. To further define these probes as
PLC substrates for future development of PLC sensors, we
carried out detailed kinetic studies to measure KM and Vmax of
these probes in PLC-catalyzed reactions. For comparison,
enzyme kinetics using PIP2 as the substrate were quantified as
The fluorescent PIP2 derivative 1 has key structural features
of endogenous PIP2 including an identical inositol phosphate
headgroup and a diacylglycerol side chain. Derivative 1 and its
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Figure 3. Kinetic studies of probes 1−3 or endogenous PIP2 with PLC-δ1. (A) The initial velocities of hydrolysis of 1, 2, or 3 at various
concentrations were fitted to the Michaelis−Menten equation. (B) Hydrolysis rates of PIP2 at various concentrations were fitted to the Michaelis−
Menten equation. (C) KM and Vmax of probes 1−3 and endogenous PIP2 with PLC-δ1.
previously described43 by varying the bulk concentration of
[3H]PIP2-containing vesicles.
mixture and sensitive detection for their capacity as substrates
of PLC-δ1. These modifications at the 6-OH group result in
derivatives that are excellent PLC substrates with marginally
lower Vmax and higher KM values compared to the unmodified
parent PIP2 derivative and endogenous PIP2. These results
demonstrate that the 6-OH group can be modified for new
functions, setting the stage for further development of sensors
selective for PLC isozymes and useful for monitoring the
spatiotemporal dynamics of these enzymes in living cells.
To increase the efficiency and accuracy of the kinetic
measurements, we carried out the experiments with probes 1−3
in a 96-well plate (Figure S1). At the indicated time, the
samples were taken from reaction mixtures with a multichannel
pipet and loaded directly onto the TLC plates. The fluorescent
components in the reaction mixture were then separated and
quantified by fluorescence. All the measurements were under
initial rate conditions (Figures S2). KM and Vmax were then
calculated by fitting the data to the Michaelis−Menten equation
(Figure 3A,C). The KM for probe 1 was 39 4.0 μM with a
Vmax of 94 0.9 pmol/(ng min) while the KM for probe 2 was
67 9.6 μM with a Vmax of 32 1.8 pmol/(ng min). Capping
of the 6-OH with a propyl group decreased Vmax and increased
KM, suggesting that the modification at 6-OH might interfere
with the key interactions of other substitutions in the inositol
headgroup with PLC-δ1. In addition, the propyl group may
increase the steric hindrance around the 1-phosphate, making
the PLC-catalyzed nucleophilic addition of the 2-OH to the 1-
phosphate a slower process. Interestingly, relative to probe 2,
probe 3 further increased KM (128 15.3 μM) while having a
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures and NMR spectra of key compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail qszhang@unc.edu; Tel 919-966-9687.
Funding
This work was funded by National Institutes of Health
(GM098894 and GM057391).
less detrimental effect on Vmax (67
9.6 pmol/(ng min)),
suggesting that increasing the length of the substitution at the
6-OH disrupts interactions with PLC-δ1 while the terminal
hydroxy of the substitution may participate in substrate-assisted
catalysis to facilitate phospholipase activity. In both cases, the
KM and Vmax of 2 and 3 are within a 3-fold range of equivalent
values for probe 1, which are similar to those for the
endogenous PIP2 (Figure 3B,C). Consequently, modifying
the 6-OH generate functional PIP2 derivatives that potentially
can be applied to monitor PLC activity spatiotemporally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Stephanie Hicks for providing purified PLC-δ1
and Dr. Weigang Huang for helpful discussions.
ABBREVIATIONS
■
PLC, phospholipase C; PIP2, phosphatidylinositol 4,5-bi-
sphosphate; IP3, inositol 1,4,5-trisphosphate; DAG, diacylgly-
cerol.
CONCLUSION
■
In conclusion, we have developed an efficient synthesis to
prepare PIP2 derivatives modified at the 6-hydroxy group,
making it possible to explore the role of the 6-OH in PIP2 for
PLC-catalyzed hydrolysis. Several of these derivatives have been
characterized in a new TLC-based assay that features
straightforward separation of products from the reaction
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